Objective To construct vectors that express phosphatidylinositol-3-kinase, catalytic, beta polypeptide （PIK3cb） shRNA in eukaryon plasmid catalyzed by PI3K in rat, then test their effects on intimal hyperplasia in transplanted vein graft. Methods One hundred and fifty SD rats were randomly divided into six groups （n=25, in each group）: blank (25% Pluronic F-127), shRNA-1, shRNA-2, 1/2 （shRNA-1+shRNA-2）, negative control (pGenesil-1 scramble shRNA) and positive control （wortmannin） group. The jugular vein in rats were interpositioned autologously into the common carotid artery. shRNA and 25% Pluronic F-127 were mixed and coated around the transplanted vein in three PIK3cb shRNA groups. Every 5 samples were removed according to the time point (1, 3, 7, 14 and 28 days after operation), respectively. The thickness of intima and neointima area were calculated and analyzed by computer system. The PCNA expression was detected by Western blot and SP immunohistochemistry. Results The intimal thickness of three PIK3cb shRNA groups were lower than those in the blank group and negative control group on day 3, 7, 14, 28 after operation (P＜0.05); The neointima area in three PIK3cb shRNA groups (except shRNA-2 group on day 3, 7) began to decrease significantly from day one (P＜0.05). The protein expression of PCNA in three PIK3cb shRNA groups on day 3 after operation were decreased compared with blank group and negative group (P＜0.05). The percentage of the PCNA positive cells area in three PIK3cb shRNA groups were significantly lower than those in blank group and negative control group in each time point (Plt;0.05). There were no significant differences between blank and negative control group in different time points (Pgt;0.05). Conclusion The PIK3cb shRNA can effectively inhibit the proliferation of vascular smooth muscle cell, which may provide a new gene therapy for the prevention of vein graft restenosis after bypass grafting.
【Abstract】 Objective To reduce restenosis in vein grafts after coronary artery bypass grafting, to investigate theeffect of human tissue factor pathway inhibitor(TFPI) gene del ivery on neointima formation. Methods The eukaryotic expressed plasmid vector pCMV-(Kozak) TFPI was constructed. Forty-eight Japanese white rabbits were randomly divided into 3 groups with 16 rabbits in each group: TFPI group, empty plasmid control group and empty control group. Animal model of common carotid artery bypass grafting was constructed. Before anastomosis, vein endothel iocytes were transfected with cationic l iposome containing the plasmid pCMV- (Kozak) TFPI (400 μg) by pressurizing infusion (30 min) in TFPI group. In empty plasmid control group, vector pCMV- (Kozak) TFPI was replaced by empty plasmid pCMV (400 μg). In empty control group, those endothel iocytes were not interfered. After operation, vein grafts were harvested at 3 days for immunohistochemical, RTPCR and Western-blot analyses of exogenous gene expression and at 30 days for histopathology measurement of intimal areas, media areas and calculation of intimal/media areas ratio. Luminal diameter and vessel wall thickness were also measured byvessel Doppler ultrasonography and cellular category of neointima was analyzed by transmission electron microscope at 30 days after operation. Results Human TFPI mRNA and protein were detected in TFPI group. The mean luminal diameter of the TFPI group, empty plasmid control group and empty control group was (2.68 ± 0.32) mm, (2.41 ± 0.23) mm and (2.38 ± 0.21) mm respectively. There were statistically significant differences between TFPI group and control groups (P lt; 0.05). The vessel wall thickness of the TFPI group, empty plasmid control group and empty control group was (1.09 ± 0.11) mm, (1.28 ± 0.16) mm and (1.34 ± 0.14) mm respectively. There were statistically significant differences between TFPI group and other control groups (P lt; 0.01). The mean intimal areas, the ratio of the intimal/media areas of the TFPI group were (0.62 ± 0.05) mm2and 0.51 ± 0.08 respectively, which were reduced compared with those of the two control groups(P lt; 0.05). The mean media areas had no significant differences among three groups (P gt; 0.05). Through transmission electron microscope analyses, no smoothmuscle cells were seen in neointima of TFPI group in many visual fields, but smooth muscle cells were found in neointima of two control groups. Conclusion Human TFPI gene transfection reduced intimal thickness in vein grafts.
Abstract:Objective To evaluate the effect of external stents on preventing vein graft neointima formation and medial thickening with non-restrictive macro porous polyester stent around porcine vein grafts. Methods Studies were performed by using ＂white race＂ pigs （n= 10） weight 25-30 kg. All the animals underwent bilateral saphenous vein into carotid artery bypass grafting. In each animal, a maeroporous stent was placed around a graft on one side and a control （unstented） graft on the opposite side. The polyester stent was shaped to cover both anastomoses completely. The size of the stem allowed unrestricted expansion of the graft in initial response to arterial pressure. After 35 days of surgery,all animals were taken to remove the grafts. Graft wall dimensions, platelet- derived growth factor （PDGF） expression and cell proliferation using proliferating cell nuclear antigen （PCNA） were measured on histological sections. Results Stents significantly reduced neointimal thickening （0. 4872 ± 0. 0706 mm vs. 0. 2259± 0. 0553mm,P〈0. 01）and medial thickening （0. 6246±0. 0859mm vs. 0. 4201±0. 0615mm,P〈0. 01）. Stents significantly reduced the percentage of cells expressing PDGF and PCNA. Media, intimal PCNA index was reduced from 7. 980/00± 4. 060/00 to 3.35±0.95%（P〈0.01）, PDGF index was reduced from 9.47%±5.35% to 2.67%± 0.97% （P 〈0. 01）. Conclusion External non-restrictive polyester stent can significantly inhibit neointimal formation and medial thickening, and may prevent late vein grafts restenosis.
Objective To study the changes of endothelin (ET) and nitric oxide (NO) in the local site of vein transfer with delayed breaking pedicle and the relation with vasospasm and vein transfer in rabbits. MethodsThe ET concentration of blood was determined with the radioimmunoassay method. The plasma NO-2,NO-3 levels in the local site of vein transfer with delayed breaking pedicle, which reflected NO levels indirectly, were detected with Ultravioletvisible (UvVIS ) spectrophotometer. ResultsThe endothelin concentration of blood was increased significantly at 2, 4 hour after the operation (P＜0.01), and at 8 hour after the operation (P＜0.05). The plasma NO level was significantly decreased at 2, 4 hour after the operation (P＜0.01). But at 24 hour after the operation, the plasma NO level was increased significantly (P＜0.05). Conclusion The recovery of ET concentration of blood and the increase of plasma NO at 24 hour after the operation are the cause of the reduced incidence of vascular crisis of vein transfer with delayed breaking pedicle, and the very time point is the optimum moment for pedicle breaking.
Objective To investigate the effect of adenovirus vector mediated transfer of human herpes simplex virus thymidine kinase (HSVtk) gene inhibits intimal hyperplasia of vein grafts. Methods Auto vein graft models of Wistar rats were established. Adenovirus vector dwelled in cervical veins which were transplanted into inferior renal abdominal aorta. The combination of HSVtk (4×109 plaque forming units) and ganciclovir (GCV) was applied to test the inhibition effect. GCV was infused 〔60 mg/(kg·d), IP, Bid〕 from day 3 to day 21 after transplantation. Vein samples were harvested and the existence of HSVtk DNA was measured by PCR and the mRNA of it was studied by in situ hybridization. Van gieson (VG) and proliferating cell nuclear antigen (PCNA) stains were carried out in paraffin sections to study the thickness of neointima and smooth muscle cells (SMCs) proliferation with a computer-assisted analysis system. The apoptosis of SMCs also was detected by TUNEL. Results The existence of HSVtk gene in veins and its transcription were demonstrated. Morphometric analysis demonstrated a reduced intima thickness in the group receiving combination therapy (HSVtk/GCV) compared with HSVtk alone 〔(17.2±3.2) μm versus (31.1±2.5) μm, P＜0.05〕. GCV per se had no effect on intimal hyperplasia after vein transplantation. The apoptosis of SMCs increased significantly and expression of PCNA decreased in HSVtk/GCV gene therapy group versus blank control group 〔(9.1±2.3)% vs (28.7±3.6)%, P＜0.05; (38.7±5.6)%vs (18.5±2.6)%, P＜0.05〕. Conclusion GCV conditions reduction of intimal hyperplasia after intraluminal delivery of HSVtk in transplanting vena veins involving SMCs apoptosis.
Objective To investigate the effects of Rho-kinase inhibitor——fasudil hydrochloride hydrate on vein graft intimal hyperplasia in vivo. Methods Twenty-four healthy rabbits (2.3-2.5 kg) were randomly divided into two groups(n=12). Fasudil hydrochloride hydrate (experimental group) and normal sodium (control group) were given 3 days beforeoperation with 30 mg/kg by intravenous injection everyday and continued until the end of the experiment. After a longitudinal incision, the femoral vein and the famoral artery were exposed about 3 cm. An approximately 2.5 cm segment of the famoral vein was harvested for the reversed-vein graft. The femoral artery was removed 1 cm segment and replaced by the harvested femoral vein. At 2 and 4 weeks after operation, the grafts were stained with HE to observe the thickness of the intima. Furthermore, the prol iferating cell nuclear antigen (PCNA) and transmission electron microscope was used to study the prol iferation of smooth muscle cell. In situ apoptosis was detected by TUNEL assay. Results All rabbits survived till the end of the experiment. The color Doppler imaging examination showed that all grafts were patency. At 2 and 4 weeks after the operation, HE staining showed that the intimal hyperplasia were obvious in the two groups. There were lots of cells in the intima, and more fusiform smooth muscle cells in the media. At 2 and 4 weeks, the intimal thickness were (30.33 ± 3.23) μm and (43.11 ± 4.92) μm in experimental group and were (44.83 ± 3.53) μm and (66.16 ± 8.45) μm in control group. The rates of PCNA positive cell were 14.28% ± 2.76% and 7.61% ± 1.06% in experimental group and were 20.08% ± 3.56% and 8.73% ± 1.35% in control group. The rates of TUNEL positive cell were 3.55% ± 0.36% and 1.22% ± 0.18% in experimental group and were 1.11% ± 0.31% and 0.55% ± 0.11% in control group. There were significant differences (P lt; 0.05) between the two groups at 2 weeks or 4 weeks, between2 weeks and 4 weeks within group. Conclusion Intravenous injection of fasudil hydrochloride hydrate is an effective method for prevention of vein graft intimal hyperplasia of rabbit.
Objective To study the mechanism of restenosis of the vein graft and the effect of the grafting injury to the vein graft. Methods One side of the 36 healthy rabbits was randomly chosen as the V-A group, and on the side a 1.5cmlong femoral vein was obtained, and an 0.5-cm-long segment of the obtained femoral vein was separated as the control group. The remaining 1-cm-long femoral vein was inverted and was autogenously implanted into the femoral artery on the same side of the rabbit. The other side of the rabbits was chosen as the V-V group, and on this side a 1-cm-long femoral vein was obtained ex vivo and then was sutured in situ. The vein grafts on both sides were harvested 4 weeks after operation. The specimens from the harvested vein grafts were stained with HE and theelastic fiber Victoria blue for an observation on the histological changes in the walls of the vein grafts, and the specimens were also stained by the immunohistochemistry of the proliferating cell nuclear antigen (PCNA) for an observation on the wall cell proliferation of the vein grafts. The changes in the ultrastructure of the proliferated wall cells of the vein grafts were observed under electron microscope. The two sides of the rabbits were compared. Results The smooth muscle cells of the media developed hyperplasia, but theintima and the media remained unchanged in their thickness (3.50±0.41 μm, 12.23±1.59 μm) in the V-V group, with no difference when compared with the control group (3.40±0.37 μm, 12.14±1.62 μm); however, when compared with the V-A group (25.60±3.21 μm, 21.30±2.47 μm),there was a significant difference in the thickness (Plt;0.01). There were no cells positive for PCNA by the immunohistochemistry examination in the control group. The cells positive for PCNA were found in the intima and the media in both the V-V group and the V-A group; however, the percentageof the cells positive for PCNA in the intima and the media was significantly greater in the V-A group than in the V-V group (16.4%±1.9% and 36.5%±3.7% vs 5.9%±1.3% and 23.4%±3.4%, Plt;0.01). In the V-V group, the endothelial cell could be observed under transmis-sion electron microscope, which was flat and had a processlike villus at its free end, and the endothelial cells were closely arranged andhad hyperplasia of the smooth muscle cells in the media. But in the V-A group,the endothelial cells had an obvious hyperplasia with an irregular shape and a widened space between the cells, and in the intima a great amount of the smooth muscle cells could be observed, which had a broken basement membrane. The smooth muscle cells also had an obvious hyperplasia in the media. The shape and alignment of the endothelial cells in the control group were similar to those in the V-V group, but the hyperplasia of the smooth muscle cells was not observed in the media. Conclusion The grafting injury can cause hyperplasia ofthe vascular wall cells, and if the hemodynamics is changed simultaneously, more serious hyperplasia and cell migration can be observed from the media to the intima, resultingin restenosis of the blood vessels. So, if we can reduce the grafting injury and improve the microcirculation of the vein graft, we may find out the methods ofpreventing restenosis of the vein graft. The animal model of the V-V graftcan help to understand the mechanism of restenosis of the vein graft.
In order to solve the defect of blood vessel in tissue transplantation and complicated palmar amputation, bridge by "Y" type vein had been used from Jan. 1990 to Jul. 1996. Twenty-three cases were treated. In this series, there were 16 males and 7 females, with ages ranged from 10 to 42 years old. Six cases were the defect of lower legs anterior skin and tibia, 3 cases were the femur fracture with injury of femoral artery and tissue’s defect, 2 cases were defect of five fingers, 12 cases were complicated palmar amputation. RESULT: 15 cases with tissue transplantation and 12 cases with limb replantation were all survival without infection or necrosis. After the following-up for 3 years (ranged from 1 to 5 years), the function of injured limbs were satisfactory, 19 patients had resumed their original work. So, to bridge by "Y" type vein is a good method for repairing the defect of blood vessels in tissue transplantation and complicated palmar amputation, but skilled microsurgery technique is required.
Objective To investigate the results and applicationvalue of crotch-shaped vein grafts in repairing defects of the vessels with a large diameter.Methods From June 1998 to October 2003, 35 cases of vesseldefects with a large diameter were repaired with crotch-shaped vein graft (29 males and 6 females,aged 18 to 45 years with an average of 25.7 years ). The locations of defects were femoral artery in 25 cases, popliteal artery in 2 cases, femoral vein in 7 cases, and subclavian vein in 1 case. The interval between injure and operatioinwas 1-8.5 hours (4.1 hours on average).The blood flows of trouble and healthy vascular were determined with Doppler detector and compared preoperatively andpostoperatively. Results All the anastomotic stomas were patent in 35 cases. Thirty-one cases were followed up 6 weeks to 24 months (9.5 months on average), the patent rate was 100%, no case occurred vasospasm or tromboembolism; 2 cases occurred stomal leak and became hematoma, 3 cases occurred muscular necrosis, and the 5 cases achieved primary healing after secondary operation. The Doppler results showed that there was statistically significant difference in the blood flow betweenpostoperation and preoperation (Plt;0.01), but no statistically significant difference when compared the trouble vascular after operation with healthy vascular (Pgt;0.05). Conclusion The methodof crotch-shaped vein grafts is safe and effective in repairing defects of vessels with a large diameter,which is easy to draw materials and handy to operate. It has a promising value in clinical application.
ObjectiveTo detect the inhibitory effect of early growth response gene-1 DNA enzyme (EDRz) on proliferation of vascular smooth muscle cell (VSMC) and intimal hyperplasia, and confirm the effect of gene therapy on stenosis and occlusion after vein transplantation. MethodsEDRz was constructed, and autogenous vein graft model was established with Wistar rats, transplanting the right jugular vein to infra renal abdominal aorta by microsurgical technique. EDRz was transfected to the graft veins and the vein graft samples were harvested at hour 1, 2, 6, 24 and on day 3, 7, 14, 28, 42 after grafting, 10 Wistar rats were randomly selected in every time. The expression of EDRz in transfected vein graft was detected by fluorescent microscope. Egr-1 mRNA was measured by reverse transcription-PCR (RT-PCR) and in situ hybridization, respectively. The protein expression of Egr-1 was detected by Western blot and immunohistochemistry, respectively. HE stained vein grafts were observed under microscope. Results① The results of EDRz transfected vein graft: At hour 1 after grafting, EDRz was mainly located in adventitia, tunica media, and partial endothelial cells of vein graft; At hour 2, 6, and 24, EDRz was located in tunica media of vein graft; and on day 7, it was mainly located in intima of vein graft. There wasn’t EDRz in vein grafts on day 14, 28, and 42. ② The results of expression of Egr-1 mRNA: Detection by RT-PCR: At hour 1 after transfecting, the expression of Egr-1 mRNA arrived at the peak, and declined at hour 2, 6, and 24. The expression was tenuity on day 3. Egr-1 mRNA expression was not found on day 7, 14, 28, and 42. The expression of Egr-1 mRNA at hour 1 was significantly higher than that of the other time point (Plt;0.01). The result of in situ hybridization was coincident with RT-PCR. ③ The results of expression of Egr-1 protein: The result of Western blot: There was no expression of Egr-1 protein in normal veins. At hour 2 after grafting, expression of Egr-1 protein was found, and declined at hour 6, 24, and on day 3. There was no expression of Egr-1 protein at hour 1, and on day 7, 14, 28, and 42. The expression of Egr-1 protein at hour 2 was significantly higher than that of the other time point (Plt;0.01). The result of immunohistochemistry was coincident with Western blot. ④The degree of VSMC hyperplasia and intimal thickness were lighter in EDRz transfected vein grafts than that in nottransfected vein grafts contemporarily. ConclusionsEDRz could reduce the expression of Egr-1 in autogenous vein graft, and could effectively restrain VSMC proliferation and intimal hyperplasia, and prevent vascular stenosis and occlusion after vein grafting.
